5-Methyl-cytosine stabilizes DNA but hinders DNA hybridization revealed by magnetic tweezers and simulations

Abstract 5-Methyl-cytosine (5mC) is one of the most important DNA modifications and plays versatile biological roles. It is well known that 5mC stabilizes DNA duplexes. However, it remains unclear how 5mC affects the kinetics of DNA melting and hybridization. Here, we studied the kinetics of unzipping and rezipping using a 502-bp DNA hairpin by single-molecule magnetic tweezers. Under constant loading rates, 5mC increases the unzipping force but counterintuitively decreases the rezipping force at various salt and temperature conditions. Under constant forces, the non-methylated DNA hops between metastable states during unzipping and rezipping, which implies low energy barriers. Surprisingly, the 5mC DNA can’t rezip after fully unzipping unless much lower forces are applied, where it rezips stochastically in a one-step manner, which implies 5mC kinetically hinders DNA hybridization and high energy barriers in DNA hybridization. All-atom molecular dynamics simulations reveal that the 5mC kinetically hinders DNA hybridization due to steric effects rather than electrostatic effects caused by the additional methyl groups of cytosines. Considering the possible high speed of DNA unzipping and zipping during replication and transcription, our findings provide new insights into the biological roles of 5mC.


Section1. Preparation of DNA for MT experiments.
As shown in Figure 1 in the main text, we prepared the DNA hairpin sample by following steps.
(ii) We digested the four dsDNA segments to generate the 4-nt sticky ends using BsaI-HF (New England Biolabs).
(iii) We ligated the four dsDNA segments to generate a 1424-bp combined dsDNA using T4 DNA ligase (New England Biolabs).
(iv) We added biotin groups to 3' ends of the dsDNA using Terminal transferase (New England Biolabs) and biotin-11-dUTP (Roche).
(v) We anchored the thiol-labelled end of dsDNA to the sulfo-SMCC (Thermo Fisher) coated glass surface.
Then, we tethered a streptavidin-coated superparamagnetic microbead Dynabeads) to the other end of dsDNA through the biotin-streptavidin bond. We exerted a stretching force of ~40 pN on the dsDNA using magnetic tweezers.
(vi) We peeled off the untethered ssDNA strand in the dsDNA at 100 mM NaOH and left the tethered ssDNA stretched at ~40 pN.
(vii) Because the two hairpin stems contained complementary sequences, the long DNA hairpin zipped at 8 pN when we changed the buffer into 1 mM Tris-HCl pH7.5 and 150 mM NaCl.
For the other DNA sample used in Figure S9, S10 and S12B, we amplified the head-to-head two 502-bp dsDNA segments (40701-41202 bp of λ-DNA) as the new hairpin stem through PCR using 2_hairpinF2-BsaI/2_hairpinR2_BsaI and 2_hairpinF3_BsaI/2_hairpinR3_BsaI as primers, respectively. We prepared this DNA sample by above steps except changed the sequence of the long hairpin.
We synthesized all the oligos with the following sequences (Sangon Biotech).  Figure S1. Home-built MT and flow cell. We built the MT using an inverted microscope equipped with an oil objective, on the top of which a flow cell was placed. We controlled the temperature with an objective heater.
To assemble one flow cell, we stuck together two cover slides with doubles-side taps and draw the inlet and outlet with silicone rubber.      Figure S13. The effects of 5mC on the unzipping and rezipping forces obtained at 150 mM NaCl using MD simulations. We stretched the 2-bp DNA hairpin through a force-cycle containing a force-increasing scan through a set of constant forces where the hairpin unzipped, followed by a force-decreasing scan through the same set of constant forces where the hairpin rezipped. At each constant force, we held the DNA for 150 ns during which we calculated the average in the extension of DNA in the last 50 ns. The force-cycle yielded an unzipping force-extension curve (filled symbols) and a rezipping force-extension curve (empty squares symbols).
We determined the unzipping force to be the lowest force where an abrupt increase in extension occurred in the unzipping force-extension curve (upward arrow). Similarly, we determined the rezipping force to be the highest force where an abrupt decrease in extension occurred in the rezipping force-extension curve (downward arrow).